Porperties of an α-bungarotoxin-binding cholinergic nicotinic receptor from drosophila melanogaster

α-[¹²⁵I]Bungarotoxin specifically binds to homogenates of Drosophila melanogaster head at levels of 0.3–0.8 pmol/mg protein. The dissociation constant calculated from rates of association and dissociation of toxin · receptor complex, is 0.6 · 10^?9M. Ca²⁺, and to lesser extent Na⁺, inhibit the reaction. α-[¹²⁵I]Bungarotoxin binding is inhibited by low concentrations of unlabelled toxin, nicotinic ligands and eserine, but not by low concentrations of muscarinic ligands, decamethonium or an organophosphate. The receptor is membrane bound and can be partially released into 100 000 × g supernatant by a combination of 1 M NaCl and 1% Triton X-100. Most of the activity in the supernatant sediments after further centrifugation at 200 000 × g for 2 h. Toxin binding sites are distinct from acetylcholinesterase molecules as revealed by pharmacological, biochemical and genetic techniques. The gene for the toxin-binding nicotinic receptor in Drosophila is apparently not located adjacent to the gene for acetylcholinesterase.     

The Mechanism of Muscarinic Receptor-Stimulated Phosphatidylinositol Resynthesis in 1321N1 Astrocytoma Cells and Its Inhibition by Li+

Abstract: The coupling of muscarinic receptor-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C to resynthesis of phosphatidylinositol (PtdIns) and the ability of Li⁺ to inhibit this after cellular inositol depletion were studied in 1321N1 astrocytoma cells cultured in medium ± inositol (40 µM). In inositol-replete cells, 1 mM carbachol/10 mM LiCl evoked an initial (0–30 min) ～≥20-fold activation of phospholipase C, whereas prolonged (>60 min) stimulation turned over Ptdlns equal to the cellular total mass, involving ～80% of the cellular Ptdlns pool without reducing PtdIns concentrations significantly. PtdIns resynthesis was achieved by a similar, initial agonist activation of PtdIns synthase. The dose dependency for carbachol stimulation of PtdIns synthase and phospholipase C was similar (EC₅₀～ 20 µM) as was the relative intrinsic activity of muscarinic receptor partial agonists. This demonstrates the tight coupling of phosphoinositide hydrolysis to resynthesis and suggests this is achieved by a direct mechanism. In inositol-replete or depleted cells basal concentrations of inositol and CMP-phosphatidate were respectively ～20 mM or ≤100–500 µM and ～0.1 or ～≥1–10 pmol/mg of protein. Comparison of the effects of agonist ± Li⁺ on the concentrations of these cosubstrates for PtdIns synthase suggest that accelerated activity of this enzyme is differentially driven by stimulated increases in the amounts of CMP-phosphatidate or inositol in inositol-replete or depleted cells, respectively. Thus, the preferential capacity of Li⁺ to impair stimulated phosphoinositide turnover in systems expressing low cellular inositol can be attributed to its ability to attenuate the stimulated rise in inositol concentrations on which such systems selectively depend to trigger accelerated PtdIns resynthesis.     

Microbial Diversity in a Pyrite-Rich Tailings Impoundment (Carnoulès,France)

The microbial communities have been investigated in the subsurface waters of the Carnoulès pyrite-rich tailings impoundment (France) for two hydrological situations characterized by the presence of oxygenated waters during winter and suboxic conditions in early autumn. In these acidic waters (2–5) characterized by elevated concentrations of Fe (1608–3354 mg · l^?1), As (130–434 mg · l^?1) and sulfates (5796–14318 mg · l^?1) and variable dissolved oxygen content, the cultivable bacteria found in these system are Thiomonas and Acidithiobacillus ferrooxidans. Molecular methods, Terminal-Restriction Fragment Length Polymorphism (T-RFLP), and 16S rRNA encoding gene library analysis indicate low diversity. The environment is dominated by only a few types of microorganisms, with 70–80% of the whole bacterial population assigned to two or three Terminal-Restriction Fragments (T-RFs). Most of these organisms are uncultured, newly described, or recently associated with acid mine drainage. Modifications of the community structure are observed as a function of the sampling period and seem to be related to the aqueous chemistry of the tailings water. At low Dissolved Oxygen (DO = 1 mg · l^?1) concentrations and moderately acidic conditions (pH = 5.7), the dominant organisms are related to the uncultured clone BA31 affiliated with Desulfosarcina variabilis, a sulfate-reducing bacteria (SRB), Acidithiobacillus ferrooxidans and the uncultured clone BVB20, closely related to Thiobacillus. At high (12 mg · l^?1) DO concentrations and low (< 2) pH values, the microbial diversity is less important and 65% of the population is assigned to the uncultured bacterium clone AS6 related to Desulfosarcina variabilis.     

Agonist stimulation at human μ opioid receptors in a [35S]GTPγS incorporation assay: observation of “bell-shaped” concentration–response relationships under conditions of strong receptor G protein coupling

Context: The appearance of “bell”- (or “inverted U”-) shaped agonist concentration–response curves (CRCs) in in vitro pharmacological experiments is a frequently observed but poorly communicated phenomenon. In the context of G protein coupled receptor research, it is commonly attributed to the recruitment of secondary targets or to desensitization or feedback processes, but the concrete background of these observations often remains intriguing. Objective: Here, we addressed the subject of bell-shaped agonist CRCs at the µ opioid receptor (µOR) by testing the impact of experimental conditions favoring G protein coupling. Methods: G protein activation by recombinant human µORs heterologously expressed in CHO cells was assessed in [³⁵S]GTPγS binding assays using the opioid ligands DAMGO, morphine, fentanyl and naloxone. Experimental conditions were varied by changing the NaCl (10–300?mM) and the GDP concentration (0.3–30?µM). Results: Both the sodium and the GDP concentration were inversely related to G protein coupling, as evident by an increase in basal [³⁵S]GTPγS incorporation at low sodium and low GDP levels and by the concomitant appearance of the partial agonist activity of the µOR antagonist, naloxone. Bell-shaped CRCs were observed for the efficacious agonists DAMGO, fentanyl and morphine, and this phenomenon was promoted by low sodium as well as by low GDP concentrations. Conclusion: µOR agonist CRCs show a non-monotonic behavior with a decline of maximal stimulation under conditions of strong receptor-G protein coupling, and this behavior is visible at the level of G protein activation itself.     

Muscarinic acetylcholine receptor in chick limb bud during morphogenesis

Summary In the chick embryo a cholinesterase activity appears in various organ anlagen which has been correlated with morphogenetic movements (Drews 1975). The cholinesterase activity is present in the mesenchyme of the limb bud during aggregation of the central chondrogenic core. In the present study binding of tritium labelled quinuclidinyl benzilate ((³H)QNB), a muscarinic antagonist, to homogenates of chick limb buds was investigated by a filtration assay. In the homogenate of limb buds at Stage 24 specific binding of (³H)QNB was demonstrated. Determination of binding constants and inhibition of binding by agonists and antagonists was studied at Stage 25/26. Specific binding was defined by the difference in binding in the absence and presence of atropine (1 M). Specific binding of (³H)QNB reflected a muscarinic receptor. The K_d in two experiments was 0.11 nM and 0.16 nM, the binding capacity was 15.7 fmol (³H)QNB/mg protein and 12.0 fmol (³H)QNB/mg protein, respectively. Data on displacement of specific bound (³H)QNB by various nicotinic and muscarinic ligands confirmed the muscarinic nature of the receptor. Muscarinic ligands inhibited the (³H) QNB binding, whereas nicotinic ligands caused no inhibition at pharmacological concentrations. I conclude that a specific muscarinic acetylcholine receptor is part of the cholinergic system whose presence is indicated by cholinesterase activity in the chondrogenic core of the limb bud during morphogenesis.     

Inhibition of Real-Time PCR in DNA Extracts from Aquifer Sediment

Variation in inhibition of real-time PCR was investigated with DNA extracts from 50 aquifer sediment core samples of 5 cm length collected through a 2.5 meter vertical profile across a landfill leachate plume. The inhibition was quantified using an internal control of the green fluorescent protein ( gfp ) gene, which was spiked into the real-time PCR reactions. The inhibition was investigated at two gfp gene concentrations: at 1.7 · 10 ⁷ gfp gene copies/g sediment (5.1 · 10 ⁴ gfp gene copies/PCR reaction) and at 1.7 · 10 ⁵ gfp gene copies/g sediment (5.1 · 10 ² gfp gene copies/PCR reaction). Despite the low TOC content of the sediment (average 0.4 mg C/g dw) the average real-time PCR response was partially inhibited, compared to a reference (pure water), at both high and low gfp concentrations. The relative amplification (reference = 1) was 0.85 ± 0.20 (high) and 0.66 ± 0.23 (low), showing significantly (P < 0.05) stronger inhibition at the lower target gene concentration. The inhibition of the real-time PCR did not show a systematic variation in the vertical profile related to plume position but variations were significant on a small scale of 5–15 cm depth intervals. One of the 50 samples failed to produce a signal with either concentration of the gfp internal control and three other samples inhibited real-time PCR at both high and low gfp concentration. These 4 samples, which were the samples with the highest inhibition, were the only DNA extracts with a visible brown colouration, indicating contents of humic-like substances. Elevated absorbance at 400 nm of these samples also indicated that humic-like substances were responsible for inhibition. However, other factors not associated with either absorbance or TOC may have contributed to the inhibition in less inhibited samples since the variation in real-time PCR response could not be sufficiently explained by absorbance or TOC. The results of this study suggest that an internal control is needed in real-time PCR reactions with DNA from environmental samples due to variation in inhibition to correctly quantify the number of target genes, especially at low target gene concentrations, when dilution of DNA extracts is not practical.     

Modulation of transmitter release from the locust forewing stretch receptor neuron by GABAergic interneurons activated via muscarinic receptors

The role of muscarinic receptors in the down‐regulation of acetylcholine (ACh) release from the locust forewing stretch receptor neuron (fSR) terminals has been investigated. Electrical stimulation of the fSR evokes monosynaptic excitatory postsynaptic potentials (EPSPs) in the first basalar motoneuron (BA1), produced mainly by the activation of postsynaptic nicotinic cholinergic receptors. The general muscarinic antagonists scopolamine (10⁻⁶ M) and atropine (10⁻⁸ to 10⁻⁶ M) caused a reversible increase in the amplitude of electrically evoked EPSPs. However, scopolamine (10⁻⁶ M) caused a slight depression in the amplitude of responses to ACh pressure‐applied to the soma of BA1. These observations indicate that the EPSP amplitude enhancement is due to the blockade of muscarinic receptors on neurons presynaptic to BA1. The muscarinic receptors may be located on the fSR itself and act as autoreceptors, and/or they may be located on GABAergic interneurons which inhibit ACh release from the fSR. Electron microscopical immunocytochemistry has revealed that GABA‐immunoreactive neurons make presynaptic inputs to the fSR. The GABA antagonist picrotoxin (10⁻⁶ M) caused a reversible increase in the EPSP amplitude, which does not appear to be due to an increase in sensitivity of BA1 to ACh, as picrotoxin (10⁻⁶ M) slightly decreased ACh responses recorded from BA1. Application of scopolamine (10⁻⁶ M) to a preparation preincubated with picrotoxin did not cause the EPSP amplitude enhancement normally seen in control experiments; in fact, it caused a slight depression. This indicates that at least some of the presynaptic muscarinic receptors are located on GABAergic interneurons that modulate transmission at the fSR/BA1 synapse. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 420–431, 1999     

Alteration of Ca2+ Fluxes in Brain Microsomes by K+ and Na+: Modulation by Sulfated Polysaccharides and Trifluoperazine

Abstract: Rat brain microsomes accumulate Ca²⁺ at the expense of ATP hydrolysis. The rate of transport is not modulated by the monovalent cations K⁺, Na⁺, or Li⁺. Both the Ca²⁺ uptake and the Ca²⁺-dependent ATPase activity of microsomes are inhibited by the sulfated polysaccharides heparin, fucosylated chondroitin sulfate, and dextran sulfate. Half-maximal inhibition is observed with sulfated polysaccharide concentrations ranging from 0.5 to 8.0 µg/ml. The inhibition is antagonized by KCl and NaCl but not by LiCl. As a result, Ca²⁺ transport by the native vesicles, which in the absence of polysaccharides is not modulated by monovalent cations, becomes highly sensitive to these ions. Trifluoperazine has a dual effect on the Ca²⁺ pump of brain microsomes. At low concentrations (20–80 µM) it stimulates the rate of Ca²⁺ influx, and at concentrations >100 µM it inhibits both the Ca²⁺ uptake and the ATPase activity. The activation observed at low trifluoperazine concentrations is specific for the brain Ca²⁺-ATPase; for the Ca²⁺-ATPases found in blood platelets and in the sarcoplasmic reticulum of skeletal muscle, trifluoperazine causes only a concentration-dependent inhibition of Ca²⁺ uptake. Passive Ca²⁺ efflux from brain microsomes preloaded with Ca²⁺ is increased by trifluoperazine (50–150 µM), and this effect is potentiated by heparin (10 µg/ml), even in the presence of KCl. It is proposed that the Ca²⁺-ATPase isoform from brain microsomes is modulated differently by polysaccharides and trifluoperazine when compared with skeletal muscle and platelet isoforms.     

Life on the edge: the plankton and chemistry of Beaver Lake, an ultra-oligotrophic epishelf lake, Antarctica

1. Beaver Lake, a large epishelf lake in eastern Antarctica was sampled on two occasions during the austral summer of 2000. Two sites, one 1 km offshore and another 6 km offshore were sampled at intervals to depths of 40 and 110 m, respectively. 2. The lake is an end member of ultra‐oligotrophic lake systems with a very low carbon pool. Dissolved organic carbon concentrations ranged between 95 and 652 μg L^–1. Nutrient levels were generally low with soluble reactive phosphorus ranging from undetectable to 8.4 μg L^–1, ammonium ranged between 1.8 and 5.0 μg L^–1, nitrate from undetectable to 161 μg L^–1 and nitrite 1.1–5.3 μg L^–1. 3. Chlorophyll a concentrations (0.39–4.38 μg L^–1) showed an unusual distribution with the highest levels close to the lake bottom at the offshore site (110 m) where the phototrophic nanoflagellates (PNAN) displayed strong autofluorescence. 4. Bacterial concentrations were low, with a maximum of 7.60 × 10⁷ L^–1, as were the concentrations of heterotrophic nanoflagellates that exploit them. 5. Primary production ranged between 19.7 and 25.49 μg C L^–1 day^–1 and bacterial production from 0.32 to 1.15 μg C L^–1 day^–1. 6. In common with other continental Antarctic lakes, the system was dominated by a microbial plankton. However, a dwarf variety of the calanoid copepod, Boeckella poppei, occurred below 25 m at concentrations of 3–5 L^–1. 7. The data suggest that primary production and bacterial production were not limited by nutrient availability, but by other factors, e.g. in the case of bacterial production by organic carbon concentrations and primary production by low temperatures.     

NUTRITIONAL REQUIREMENTS OF THE CHYTRID KARLINGIA ASTEROCYSTA,AN OBLIGATE CHITINOPHILE

The nonfilamentous chytrid, Karlingia asterocysta, has been isolated in pure culture on chitin media and the nutritional requirements of a single-spore clone investigated. The fungus displayed an absolute requirement for chitin or preformed N-acetyl-d -glucosamine. This requirement could only be relieved partially by glucose in the presence of limiting acetyl glucosamine concentrations. Under similar conditions other carbohydrates were not utilized. Sulfate was used as a sulfur source and either nitrate or ammonium ion served as nitrogen sources, though growth was better with amino acids. The organism had a very low phosphate optimum (5 × 10^–5 m ) and was inhibited by concentrations at or above 1 × 10^–3 m . The optimal pH range extended from 6.0 to 7.5 and growth decreased rapidly at higher or lower pH values. Thiamine was required at a very low concentration; only 2 μg thiamine-HCl/liter were required for optimal growth. In a rich, agitated medium K. asterocysta completed a single growth cycle (i.e., plant generation) in 70 hr at 25 C.     

Cholinergic Dilatation and Constriction of Feline Cerebral Blood Vessels Are Mediated by Stimulation of Phosphoinositide Metabolism via Two Different Muscarinic Receptor Subtypes

Abstract: The muscarinic receptors involved in phosphoinositide (PI) hydrolysis have been pharmacologically characterized in cat cerebral blood vessels. Carbachol elicited a concentration-dependent increase in inositol phosphate accumulation [inositol monophosphate, bisphosphate, trisphosphate (IP₃) and tetrakisphosphate] in both major cerebral arteries and small pial vessels, which reached 140–280% of baseline at 10^?3M carbachol (referred to as maximal effect). However, the inositol phosphate accumulation response was found to be biphasic with a submaximal effect (30–50% of the maximal stimulation) obtained at low carbachol concentrations (<10^?5M). Endothelial denudation induced a virtual disappearance of the submaximal PI response without affecting that elicited by high concentrations of carbachol. The pharmacology of the two carbachol-induced PI responses was investigated by comparing the potency of selected muscarinic antagonists to block the IP₃ accumulation induced by 10^?7M (endothelium-dependent submaximal effect) and 10^?4M (endothelium-independent near-maximal effect) carbachol. In both major arteries and pial vessels, the activation of IP₃ production by 10^?4M carbachol was similarly inhibited by muscarinic antagonists with the following averaged rank order of potency (in -log IC₅₀): 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 8.65) > pirenzepine (8.28) > 6-chloro-5,10-dihydro-5-[(1-methyl-4-piperidinyl)acetyl]-11H-dibenzo[b,e][1,4]diazepine-11-one (UH-AH 371; 7.87) > 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,-11-dihydro-6H-pyridol[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116; 6.62), a pharmacological profile compatible with an M₁ receptor subtype. In contrast, the submaximal stimulation of the PI metabolism elicited by 10^?7M carbachol in major arteries was blocked by the same antagonists with the following order of potency (in -log IC₅₀): 4-DAMP (8.38) > pirenzepine (7.25) > UH-AH 371 (6.25) > AF-DX 116 (5.72), which was reminiscent of an M₃ pharmacological profile. These findings indicate that stimulation of cerebrovascular muscarinic receptors is accompanied by PI hydrolysis via two distinct receptors, most probably the M₁ and M₃ subtypes that have been associated with constriction and dilatation, respectively, of cat cerebral arteries. Furthermore, these results provide strong evidence for an endothelial localization of the M₃ dilatatory receptors within the vessel wall.     

Effects of irradiance, water temperature and nutrients on the growth of sporelings of the crustose coralline alga Lithophyllum yessoense Foslie (Corallinales, Rhodophyceae)

Lithophyllum yessoense Foslie is a markedly dominant subtidal, crustose coralline alga in south–western Hokkaido, Japan. In this study, the effects of irradiance, water temperature and nutrients (nitrate and phosphate) on the growth of sporelings of the alga were examined. The relative growth rate (RGR) was saturated at 17.6% d^?1 at a high irradiance (240 umol photon m²s^?1). Even at a low irradiance (10.7–49.9 umol photon m^?2s^?1), RGR was 7.1–12.7% d^?1 The survival rate of sporelings was greater than 80% at irradiance above 10.7 μmol photon m^?2s^?1 throughout the culture period. The growth of L. yessoense sporelings was promoted at 15°C and 20°C, but inhibited at 5°C. The half‐saturation constants (Ks) for growth were about 0.5 umol L^?1 and 0.14 umol L^?1 for nitrate and phosphate, respectively. Saturated nitrate and phosphate concentrations for the growth were about 4.0 μmol L^?1 and 0.4 μmol L^?1, respectively, suggesting that L. yessoense is adaptable to a relatively high water temperature, a wide range of irradiance, and low ambient nitrate and phosphate concentrations. The results provide a possible explanation of why L. yessoense is dominant in the environments of south‐western Hokkaido.     

Effects of manganese on potassium outflow from erythrocytes and on respiration of rat liver mitochondria

In this work, effects of manganese on respiration of rat liver mitochondria and the rate of K⁺ outflow from rat erythrocytes are studied in a broad range of concentrations. It is shown that manganese ions at low concentrations (1 × 10^–7–3 × 10^–5 М) inhibit K⁺ outflow from rat erythrocytes; this can be used to prevent their lysis. At high concentrations (1 × 10^–4–1 × 10^–3 M), manganese activates K⁺ outflow from the erythrocytes but inhibits the valinomycin-induced outflow of the ion from the erythrocytes. This fact is an indication of manganese influence on physicochemical properties of membranes. At low concentrations manganese does not affect parameters of respiration and oxidative phosphorylation of rat liver mitochondria, while at high concentrations it exerts acceleration of the mitochondrial respiration, i.e., uncouples respiration from phosphorilation and, hence, inhibits ATP synthesis.     

Effects of D-Tubocurarine on Rat Cardiac Muscarinic Receptors: A Comparison with Gallamine

Abstract

Gallamine and d-tubocurarine inhibited (³H)N-methylscopolamine ((³H)NMS) binding to rat cardiac muscarinic receptors with I₅₀ values of 0.7 μM and 22 μM, respectively. They decreased the association and dissociation rates of the two ligands (³H)NMS and (³H)Oxotremorine M ((³H)Oxo-M).

Gallamine interaction with muscarinic receptors was markedly inhibited by (³H)NMS and (³H)Oxo-M binding to the receptors. We were unable to demonstrate (³H)NMS or (³H)Oxo-M binding to the muscarinic receptor-gallamine complex.

By contrast, d-tubocurarine interaction with rat cardiac muscarinic receptors was facilitated by (³H)Oxo-M binding and only slightly inhibited by (³H)NMS binding to muscarinic binding sites. Furthermore, (³H)NMS and (³H)Oxo-M bound to the receptor-d-tubocurarine complex, indicating that the latter drug interacted with an allosteric site on cardiac muscarinic receptors but did not recognize the muscarinic binding site (at concentrations below 1 mM).     

Suspended clay reduces Daphnia feeding rate

SUMMARY. 1. Suspended sediments often reduce cladoceran abundance in the field, and reduce the algal feeding rates of cladocerans in the laboratory. This paper explores the behavioural mechanisms by which suspended clay reduces Daphnia feeding rates. Feeding experiments using radiolabelled Cryptomonas cells showed that 50–200 mg 1-^?1 coarse suspended clay (particle size<2 μm) reduced the algal ingestion rate of Daphnia ambigua by 29–87%, but fine suspended clay (<1 μm) had no effect. Suspended clay decreased feeding rate by 60–70% at low algal concentrations (≤5×10³ cells ml^?1), but by only 27% at high algal concentrations (20×10³ cells ml^?1). Thus, the inhibitory effects of suspended clay are greater at low algal concentrations. The sudden addition (or removal) of suspended clay caused immediate reductions (or increases) in algal ingestion rate. 2. Observations of the feeding behaviour of tethered D.pulex showed that the frequency of postabdominal rejections increased greatly in the presence of suspended clay. The rejected boluses contained both algae and clay. Thoracic feeding appendage beat frequency decreased in the presence of suspended clay, decreasing the volume of water searched for food particles. 3. These behavioural responses indicate that clay reduces cladoceran feeding rate by mechanically interfering with both the collection and ingestion of algal cells. Both inhibitory effects are caused because cladocerans collect and ingest suspended clay particles. The behavioural mechanisms by which cladocerans regulate their feeding rate in very high concentrations of algal cells (rejection of excess food and reduction in thoracic limb pumping movements) are the same mechanisms responsible for the inhibition of algal ingestion rate in the presence of high concentrations of suspended clay particles.     

Effect of food concentration on the production and viability of resting eggs of the rotifer Brachionus: implications for the timing of sexual reproduction

1. Life‐table experiments with Brachionus calyciflorus test several hypotheses related to the idea that sexual reproduction in monogonont rotifers should occur when food resources are favourable. 2. The food concentration necessary for a fertilised mictic female to produce one phenotypically normal resting egg was higher than that for an amictic female to produce one daughter. At the lowest concentration of Cryptomonas erosa (1.25 × 10³ cells mL^?1), the lifetime fecundity of these two types of females was 0.9 and 1.4, respectively. 3. The lifetime fecundity of both fertilised mictic females and amictic females increased with food concentration to 3.4 resting eggs and 15.2 daughters female^?1, respectively. The approach to maximal fecundity with increasing food concentration was more rapid for fertilised mictic females, such that their lifetime fecundity relative to that of amictic females gradually decreased from 0.64 (at 1.25 × 10³ C. erosa mL^?1) to 0.22 (at 2.5 × 10⁴ C. erosa mL^?1). 4. The probability of a fertilised mictic female producing one or more abnormal resting eggs during her lifetime was high (approximately 75%). The mean proportion of abnormal eggs produced per female varied among the different food‐concentration treatments (26–38%) but was not higher at the low food concentrations. 5. The proportion of normal resting eggs that hatched was high (51–71%); those produced at low food concentrations were no less likely to hatch than those produced at high food concentrations. No abnormal resting eggs hatched. 6. The probability of a fertilised mictic female producing an abnormal resting egg increased rapidly with her age at all food concentrations. The probability of a normal resting egg hatching declined with maternal age at the low food concentration in one of two experiments. 7. The results support the idea that induction of mictic females should occur when food resources are good. Coincidence of sexual reproduction with low food availability risks low production of resting eggs for several reasons. Population size may be small, with a low probability of encounters between young mictic females and males, and fertilised mictic females may be unable to mature and produce resting eggs.     

Characterization and Regulatory Properties of Glucose-6-Phosphate Dehydrogenase from Black Gram (Phaseolus mungo)

Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) was partially purified by fractionation with ammonium sulfate and phosphocellulose chromatography. The K_m value for glucose-6-phosphate is 1.6 × 10^?4 and 6.3 × 10^?4M at low (1.0–6.0 × 10^?4M) and high (6.0–30.0 × 10^?4M) concentrations of the substrate, respectively. The K_m value for NADP⁺ is 1.4 × 10^?5M. The enzyme is inhibited by NADPH, 5-phosphoribosyl-1-pyrophosphate, and ATP, and it is activated by Mg²⁺, and Mn²⁺. In the presence of NADPH, the plot of activity vs. NADP⁺ concentration gave a sigmoidal curve. Inhibition of 5-phosphoribosyl-1-pyrophosphate and ATP is reversed by Mg²⁺ or a high pH. It is suggested that black gram glucose-6-phosphate dehydrogenase is a regulatory enzyme of the pentose phosphate pathway.     

HOT WATER EXTRACTABLE PHOSPHORUS—AN INDICATOR OF NUTRITIONAL STATUS OF PERIDINIUM CINCTUM (DINOPHYCEAE) FROM LAKE KINNERET (ISRAEL)?1

The intracellular levels of hot water extractable and total phosphorus were determined in the dinoflagellate Peridinium cinctum. f. westii (Lemm.) Lef. for natural samples from the bloom in Lake Kinneret and from laboratory cultures. Amounts of phosphorus (P) in the hot water fraction, relative to total cellular phosphorus, were similar in lake Peridinium and in cells grown in high ambient orthophosphate (P_i) media (3–6 mg P · l^?1). The absolute amounts of hot water extractable P in natural cell and those cultured at lower P_i concentrations (0.02–0.05 mg P · 1^?1) were similar, although average P_i in lake water were 4 μg · l^?1. Under most growth conditions the hot water extract contained approximately equal amounts of molybdate reactive phosphorus (MRP) and non-MRP. Short chain (6–9 units) polyphosphates (mol wt 630–950) probably constituted the bulk of the non-MRP pool, which was hydrolysable by alkaline phosphatase and may serve as a precursor for a more permanent P store. Intracellular P levels and distribution were not directly dependent on external P_i concentrations but may be determined by the N:P atomic ratio or overall external ionic milieu. Peridinium grown in low ambient P_i released significant amounts of non-MRP compounds. In Lake Kinneret, for at least most of the bloom period, Peridinium does not appear to be limited by P supply.     

Increased Insulin Secretion by Muscarinic M1 and M3 Receptor Function from Rat Pancreatic Islets in vitro

Parasympathetic system plays an important role in insulin secretion from the pancreas. Cholinergic effect on pancreatic beta cells exerts primarily through muscarinic receptors. In the present study we investigated the specific role of muscarinic M1 and M3 receptors in glucose induced insulin secretion from rat pancreatic islets in vitro. The involvement of muscarinic receptors was studied using the antagonist atropine. The role of muscarinic M1 and M3 receptor subtypes was studied using subtype specific antagonists. Acetylcholine agonist, carbachol, stimulated glucose induced insulin secretion at low concentrations (10⁻⁸–10⁻⁵ M) with a maximum stimulation at 10⁻⁷ M concentration. Carbachol-stimulated insulin secretion was inhibited by atropine confirming the role of muscarinic receptors in cholinergic induced insulin secretion. Both M1 and M3 receptor antagonists blocked insulin secretion induced by carbachol. The results show that M3 receptors are functionally more prominent at 20 mM glucose concentration when compared to M1 receptors. Our studies suggest that muscarinic M1 and M3 receptors function differentially regulate glucose induced insulin secretion, which has clinical significance in glucose homeostasis.     

Nicotinic Autoreceptors Mediating Enhancement of Acetylcholine Release Become Operative in Conditions of "Impaired" Cholinergic Presynaptic Function

Abstract: The existence in the mammalian CNS of release-inhibiting muscarinic autoreceptors is well established. In contrast, few reports have focused on nicotinic autoreceptors mediating enhancement of acetylcholine (ACh) release. Moreover, it is unclear under what conditions the function of one type of autoreceptor prevails over that of the other. Rat cerebrocortex slices, prelabeled with [³H]choline, were stimulated electrically at 3 or 0.1 Hz. The release of [³H]ACh evoked at both frequencies was inhibited by oxotremorine, a muscarinic receptor agonist, and stimulated by atropine, a muscarinic antagonist. Nicotine, ineffective at 3 Hz, enhanced [³H]ACh release at 0.1 Hz; mecamylamine, a nicotinic antagonist, had no effect at 3 Hz but inhibited [³H]ACh release at 0.1 Hz. The cholinesterase inhibitor neostigmine decreased [³H]ACh release at 3 Hz but not at 0.1 Hz; in the presence of atropine, neostigmine potentiated [³H]ACh release, an effect blocked by mecamylamine. In synaptosomes depolarized with 15 mM KCI, ACh inhibited [³H]ACh release; this inhibition was reversed to an enhancement when the external [Ca²⁺] was lowered. The same occurred when, at 1.2 mM Ca²⁺, external [K⁺] was decreased. Oxotremorine still inhibited [³H]ACh release at 0.1 mM Ca²⁺. When muscarinic receptors were inactivated with atropine, the K⁺ (15 mM)-evoked release of [³H]ACh (at 0.1 mM Ca²⁺) was potently enhanced by ACh acting at nicotinic receptors (EC₅₀? 0.6 µM). In conclusion, synaptic ACh concentration does not seem to determine whether muscarinic or nicotinic autoreceptors are activated. Although muscarinic autoreceptors prevail under normal conditions, nicotinic autoreceptors appear to become responsive to endogenous ACh and to exogenous nicotinic agents under conditions mimicking impairment of ACh release. Our data may explain in part the reported efficacy of cholinesterase inhibitors (and nicotinic agonists) in Alzheimer's disease.